Biosynthesis of Betalains Elicited by Methyl Jasmonate in Two Species of Alternanthera Genus: Antagonistic Regulations Result in Increase of Pigments.

Natural pigments are components very important in the dye industry. The betalains are pigments found in plants from Caryophyllales order and are relevant in the food manufacturing. The main source of betalains is beetroot, which has unfavorable aftertaste. Therefore, the demand for alternative species producing betalains has increased. Elicitor molecules such as methyl jasmonate (MeJA) induce metabolic reprogramming acting in the biosynthesis of specialized metabolites and can enhance pigment concentrations. Here, we used this strategy to identify if treatment with MeJA at 100 µM can promote the accumulation of betalains and other bioactive compounds in Alternanthera philoxeroides and Alternanthera sessilis. We performed the gene expression, concentration of betalains, phenols, flavonoids, amino acids (phenylalanine and tyrosine), and antioxidant activity. The results showed that MeJA treatment increased betalains and other bioactive compounds in the two Alternanthera species but A. sessilis had a better performance. One key factor in this pathway is related to the phenylalanine and tyrosine concentration. However, the species have distinct metabolic regulation: in A. philoxeroides, high concentrations of betalain pigments increase the tyrosine concentration and gene expression (include ADH) under MeJA and in A. sessilis, high concentrations of betalain pigments reduce the gene expression and tyrosine concentration after 2 days under MeJA. This study brings new questions about betalain biosynthesis and sheds light on the evolution of this pathway in Caryophyllales.

Macroevolutionary trends and diversification dynamics in Atripliceae (Amaranthaceae s.l., Chenopodioideae): a first approach.

BACKGROUND AND AIMS: Atripliceae evolved and diversified by dispersals and radiations across continents in both hemispheres, colonizing similar semi-arid, saline-alkaline environments throughout the world. Meanwhile, its species developed different life forms, photosynthetic pathways, mono- or dioecy, and different morphological features in flowers, fruiting bracteoles and seeds. In this study, we introduce a first approach to the macroevolutionary patterns and diversification dynamics of the Atripliceae to understand how time, traits, speciation, extinction and new habitats influenced the evolution of this lineage. METHODS: We performed molecular phylogenetic analyses and clade age estimation of Atripliceae to apply time-, trait- and geographic-dependent diversification analyses and ancestral state reconstructions to explore diversification patterns within the tribe. KEY RESULTS: Opposite diversification dynamics within the two major clades of Atripliceae, the Archiatriplex and Atriplex clades, could explain the unbalanced species richness between them; we found low mean speciation rates in the Archiatriplex clade and one shift to higher speciation rates placed in the branch of the Atriplex core. This acceleration in diversification seems to have started before the transition between C3 and C4 metabolism and before the arrival of Atriplex in the Americas, and matches the Mid-Miocene Climatic Optimum. Besides, the American species of Atriplex exhibit slightly higher net diversification rates than the Australian and Eurasian ones. While time seems not to be associated with diversification, traits such as life form, photosynthetic pathway and plant sex may have played roles as diversification drivers. CONCLUSIONS: Traits more than time played a key role in Atripliceae diversification, and we could speculate that climate changes could have triggered speciation. The extreme arid or saline environments where Atripliceae species prevail may explain its particular evolutionary trends and trait correlations compared with other angiosperms and highlight the importance of conservation efforts needed to preserve them as genetic resources to deal with climatic changes.

Protoplast Isolation and Transfection in the Single-Cell C4 Species Bienertia sinuspersici.

We have developed an optimized protocol for isolating protoplasts from chlorenchyma cells of the single-cell C4 species Bienertia sinuspersici. The isolated protoplasts maintained the integrity of the unique single-cell C4 intracellular compartmentation of organelles as observed in chlorenchyma cells after cell wall digestion. Approximately over 80% of isolated protoplasts expressed the fusion reporter gene following the polyethylene glycol-mediated transfection procedures. Overall, fluorescent protein fusion tagged with various intraorganellular sorting signals validated the potential use of the transient gene expression system in subcellular localization and organelle dynamics studies.

Detection, Identification, and Molecular Characterization of the 16SrII-V Subgroup Phytoplasma Strain Associated with Digera muricata in Taiwan.

Digera muricata (L.) Mart. is a pantropical annual herb belonging to the Amaranthaceae family. In August 2021, D. muricata with indicative phytoplasma symptoms of phyllody, witches'-broom, and virescence was discovered adjacent to a peanut field in Mailiao, Yunlin, Taiwan. The causal agent of the observed symptoms was detected and identified by a series of molecular characterizations. Sieve elements of the phloem tissue were perused under the transmission electron microscope and revealed the presence of pleomorphic phytoplasma-like organisms. Nested PCR using phytoplasma universal primer pairs P1/P7 and R16F2n/R16R2 was able to amplify a 1.2-kb DNA fragment for the 16S rRNA gene only from the symptomatic D. muricata. The 16S rRNA-based phylogenetic analysis and the iPhyClassifier-based virtual RFLP further affirmed that the phytoplasma associated with the diseased D. muricata can be classified into the 16SrII-V subgroup. Moreover, displayed evident symptoms were explained by the concomitant detection of PHYL1 and SAP11, the virulence genes responsible for the development of leaf-like flowers and shoot proliferation, respectively. Although phytoplasma infection on the noncrop species does not have a direct economic impact, its role in disease spread and perpetuation is indubitable.

Insights into Regulation of C2 and C4 Photosynthesis in Amaranthaceae/Chenopodiaceae Using RNA-Seq.

Amaranthaceae (incl. Chenopodiaceae) shows an immense diversity of C4 syndromes. More than 15 independent origins of C4 photosynthesis, and the largest number of C4 species in eudicots signify the importance of this angiosperm lineage in C4 evolution. Here, we conduct RNA-Seq followed by comparative transcriptome analysis of three species from Camphorosmeae representing related clades with different photosynthetic types: Threlkeldia diffusa (C3), Sedobassia sedoides (C2), and Bassia prostrata (C4). Results show that B. prostrata belongs to the NADP-ME type and core genes encoding for C4 cycle are significantly upregulated when compared with Sed. sedoides and T. diffusa. Sedobassia sedoides and B. prostrata share a number of upregulated C4-related genes; however, two C4 transporters (DIT and TPT) are found significantly upregulated only in Sed. sedoides. Combined analysis of transcription factors (TFs) of the closely related lineages (Camphorosmeae and Salsoleae) revealed that no C3-specific TFs are higher in C2 species compared with C4 species; instead, the C2 species show their own set of upregulated TFs. Taken together, our study indicates that the hypothesis of the C2 photosynthesis as a proxy towards C4 photosynthesis is questionable in Sed. sedoides and more in favour of an independent evolutionary stable state.

Comparative transcriptome analysis reveals ecological adaption of cold tolerance in northward invasion of Alternanthera philoxeroides.

BACKGROUND: Alternanthera philoxeroides (alligator weed) is a highly invasive alien plant that has continuously and successfully expanded from the tropical to the temperate regions of China via asexual reproduction. During this process, the continuous decrease in temperature has been a key limiting environmental factor. RESULTS: In this study, we provide a comprehensive analysis of the cold tolerance of alligator weed via transcriptomics. The transcriptomic differences between the southernmost population and the northernmost population of China were compared at different time points of cold treatments. GO enrichment and KEGG pathway analyses showed that the alligator weed transcriptional response to cold stress is associated with genes encoding protein kinases, transcription factors, plant-pathogen interactions, plant hormone signal transduction and metabolic processes. Although members of the same gene family were often expressed in both populations, the levels of gene expression between them varied. Further ChIP experiments indicated that histone epigenetic modification changes at the candidate transcription factor gene loci are accompanied by differences in gene expression in response to cold, without variation in the coding sequences of these genes in these two populations. These results suggest that histone changes may contribute to the cold-responsive gene expression divergence between these two populations to provide the most beneficial response to chilling stimuli. CONCLUSION: We demonstrated that the major alterations in gene expression levels belonging to the main cold-resistance response processes may be responsible for the divergence in the cold resistance of these two populations. During this process, histone modifications in cold-responsive genes have the potential to drive the major alterations in cold adaption necessary for the northward expansion of alligator weed.

Plants' genetic variation approach applied to zinc contamination: secondary metabolites and enzymes of the antioxidant system in Pfaffia glomerata accessions.

Zinc (Zn) is a micronutrient, but its excessive concentration can impair plant growth and development. Fertilizers, liming materials, pesticides and fungicides containing Zn have contributed to increase its concentration in agricultural soils. The aim of the present study is to evaluate the effect of Zn excess on the non-enzymatic (anthocyanin and ß-ecdysone) and enzymatic (superoxide dismutase-SOD and guaiacol peroxidase-GPX) antioxidant system of two P. glomerata accessions (JB and GD) grown in hydroponic system and soil, under short- and long-term exposure times. Three Zn levels (2, 100 and 200 µM) and two short-term exposure times (7 and 14 d) were tested in the hydroponic experiment. Three Zn levels (2, 100 and 200 mg kg-1) and two long-term exposure times (34 and 74 d) were tested in the soil experiment. The effects of Zn excess on P. glomerata accessions depended on the growth system and exposure time. Zinc excess in both tested growth systems resulted in significant change in the tissue oxidative process (MDA concentration) in both accessions, as well as broadened the antioxidant system response, which was based on antioxidant enzymes (SOD and GPX) and secondary metabolites (anthocyanins and ß-ecdysone). The highest anthocyanin concentration was observed in accession JB, which was grown in hydroponics, but tissue anthocyanin concentration increased in both accessions, regardless of growth medium and exposure time. The ß-ecdysone concentration in the roots increased in both accessions, but accession GD was more responsive to Zn excess. There was significant physiological variation in P.glomerata accessions in response to Zn excess.

The EccDNA Replicon: A Heritable, Extranuclear Vehicle That Enables Gene Amplification and Glyphosate Resistance in Amaranthus palmeri.

Gene copy number variation is a predominant mechanism used by organisms to respond to selective pressures from the environment. This often results in unbalanced structural variations that perpetuate as adaptations to sustain life. However, the underlying mechanisms that give rise to gene proliferation are poorly understood. Here, we show a unique result of genomic plasticity in Amaranthus palmeri: a massive, ∼400-kb extrachromosomal circular DNA (eccDNA) that harbors the 5-ENOYLPYRUVYLSHIKIMATE-3-PHOSPHATE SYNTHASE (EPSPS) gene and 58 other genes whose encoded functions traverse detoxification, replication, recombination, transposition, tethering, and transport. Gene expression analysis under glyphosate stress showed transcription of 41 of these 59 genes, with high expression of EPSPS, as well as genes coding for aminotransferases, zinc finger proteins, and several uncharacterized proteins. The genomic architecture of the eccDNA replicon is composed of a complex arrangement of repeat sequences and mobile genetic elements interspersed among arrays of clustered palindromes that may be crucial for stability, DNA duplication and tethering, and/or a means of nuclear integration of the adjacent and intervening sequences. Comparative analysis of orthologous genes in grain amaranth (Amaranthus hypochondriacus) and waterhemp (Amaranthus tuberculatus) suggests that higher order chromatin interactions contribute to the genomic origins of the A. palmeri eccDNA replicon structure.

Cytogenetic analysis of Bienertia sinuspersici Akhani as the first step in genome sequencing.

BACKGROUND: C4 plants are efficient in suppressing photorespiration and enhancing carbon gain as compared to C3 plants. Bienertia sinuspersici Akhani is one of the few species in the family Amaranthaceae that can perform C4 photosynthesis within individual chlorenchyma cells, without the conventional Kranz anatomy in its leaf. This plant is salt-tolerant and is well-adapted to thrive in hot and humid climates. To date, there have been no reported cytogenetic analyses yet on this species. OBJECTIVE: This study aims to provide a cytogenetic analysis of B. sinuspersici as the first step in genome sequencing. METHODS: Fluorescence in situ hybridization (FISH) karyotype analysis was conducted using the metaphase chromosomes of B. sinuspersici probed with 5S and 45S rDNA and Arabidopsis-type telomeric repeats. RESULTS: Results of the cytogenetic analysis confirmed that B. sinuspersici has 2n = 2x = 18 consisting of nine pairs of metacentric chromosomes. Two loci of 45S rDNA were found on the distal regions of the short arm of chromosome 7. Nine loci of 5S rDNA were found in the pericentromeric regions of chromosomes 1, 3, 4, 6, and 8, which also colocalized with Arabidopsis-type telomeric repeats; while four loci in the interstitial regions of chromosome 5 and 8 can be observed. The single locus of 5S rDNA that was found in chromosome 8 appears to be hemizygous. CONCLUSION: The FISH karyotype analysis, based on the combination of rDNAs, telomeric tandem repeat markers and C0t DNA chromosome landmarks, allowed efficient chromosome identification and provided useful information in characterizing the genome of B. sinuspersici.

Endophytic association of bioactive and halotolerant Humicola fuscoatra with halophytic plants, and its capability of producing anthraquinone and anthranol derivatives.

Halophytic plants growing in harsh desert environments are rich reservoirs of unique endophytic microorganisms. Here, healthy fresh plants of the families Tamaricaceae and Amarantaceae at three saline locations in Iran were investigated for their bioactive endophytic fungi. Among a vast number of isolates, eight isolates were identified as Humicola fuscoatra (Sordariomycetes, Pezizomycotina, Ascomycota) by microscopy and representative DNA sequences of the 5.8S rDNA (ITS) and partial ß-tubulin (TUB2). Those isolates were halotolerant, and highly bioactive, so that their intra- and extra-cellular metabolites possessed in vitro antifungal, antibacterial and antiproliferative activities, against a number of fungal and bacterial plant pathogens including the fungi Arthrobotrys conoides, Pyrenophora graminea, Pyricularia grisea and the bacteria Agrobacterium tumefaciens, Pseudomonas syringae and Xanthomonas oryzae. Chemical analyses of metabolites from the endophytes using HNMR, CNMR, NOESY, COSY, HMBC, HSQC, DEPT, TOCSY and EI MASS techniques identified 3,8-dihydroxy-1-methyl-9,10-anthracenedione (aloesaponarin II; an anthraquinone derivative), 1,8,9-anthracenetriol structure (chrysarobin; an anthranol derivative) and 2,4-di-tert-butylthiophenol in fungal extracts. To the best of our knowledge, this is the first report of endophytic association of halotolerant H. fuscoatra isolates with Tamaricaceae and Amarantaceae, and their bioactivity against plant pathogens. Also, the capability of chrysarobin and aloesaponarin II production is new to the fungal kingdom. These findings may find application in agriculture, pharmacology, and biotechnology.

Studies on genome size estimation, chromosome number, gametophyte development and plant morphology of salt-tolerant halophyte Suaeda salsa.

BACKGROUND: Soil salinization and alkalization are among the major agricultural threats that affect crop productivity worldwide, which are increasing day by day with an alarming rate. In recent years, several halophytes have been investigated for their utilization in soil remediation and to decipher the mechanism of salt-tolerance in these high salt tolerant genetic repositories. Suaeda salsa is an annual halophytic herb in the family Amaranthaceae, displaying high salt and alkali-resistance and having nutritive value. However, the fundamental biological characteristics of this valuable plant remain to be elucidated until today. RESULTS: In this study, we observed the morphology and development of Suaeda salsa, including seed morphology, seed germination, plant morphology, and flower development. Using microscopy, we observed the male and female gametophyte developments of Suaeda salsa. Also, chromosome behaviour during the meiosis of male gametophyte was studied. Eventually, the genome size of Suaeda salsa was estimated through flow cytometry using Arabidopsis as reference. CONCLUSIONS: Our findings suggest that the male and female gametophyte developments of Suaeda salsa are similar to those of the model plant Arabidopsis, and the diploid Suaeda salsa contains nine pairs of chromosomes. The findings also indicate that the haploid genome of Suaeda salsa is approximately 437.5 MB. The observations and results discussed in this study will provide an insight into future research on Suaeda salsa.

Effects of parental light environment on growth and morphological responses of clonal offspring.

Environments experienced by parent ramets of clonal plants can potentially influence fitness of clonal offspring ramets. Such clonal parental effects may result from heritable epigenetic changes, such as DNA methylation, which can be removed by application of DNA de-methylation agents such as 5-azacytidine. To test whether parental shading effects occur via clonal generation and whether DNA methylation plays a role in such effects, parent plants of the clonal herb Alternanthera philoxeroides were first subjected to two levels of light intensity (high versus low) crossed with two levels of DNA de-methylation (no or with de-methylation by application of 5-azacytidine), and then clonal offspring taken from each of these four types of parent plant were subjected to the same two light levels. Parental shading effects transmitted via clonal generation decreased growth and modified morphology of clonal offspring. Offspring responses were also influenced by DNA methylation level of parent plants. For clonal offspring growing under low light, parental shading effects on growth and morphology were always negative, irrespective of the parental de-methylation treatment. For clonal offspring growing under high light, parental shading effects on offspring growth and morphology were negative when the parents were not treated with 5-azacytidine, but neutral when they were treated with 5-azacytidine. Overall, parental shading effects on clonal offspring performance of A. philoxeroides were found, and DNA methylation is likely to be involved in such effects. However, parental shading effects contributed little to the tolerance of clonal offspring to shading.

Reactive green dye remediation by Alternanthera philoxeroides in association with plant growth promoting Klebsiella sp. VITAJ23: A pot culture study.

Contamination of soil by textile effluent is a major threat found worldwide. These pollutants have diverse range of negative effects on the ecosystem, therefore restoration through cost effective biological strategy is the need of the hour. The aim of the current study was to enhance the decolourization of reactive green dye (RGD) using phytoremediation coupled with augmentation of effective bacteria to the rhizosphere. The isolate Klebsiella sp. VITAJ23 was isolated from textile effluent polluted soil and was assessed for its plant growth promoting traits (PGP) and the PGP functional genes were amplified. The soil was artificially polluted with RGD concentration ranging from 1000 to 3000 mg kg-1 and Alternanthera philoxeroides plantlets were planted in phyto and rhizoremediation treatments, the setup was maintained upto 60 d. The isolate VITAJ23 was augmented in the rhizoremediation setup and the morphological parameters were assessed at regular interval. There was a significant increase in the chlorophyll content as well as root and shoot length of the plant when treated with the bacterial suspension. Decolourization study revealed 79% removal of reactive green dye with an enhanced oxido-reductase enzyme activity in the setup bioaugmented with bacteria. The biodegraded metabolites were identified as 2-allylnapthalene, l-alanine, n-acetyl-and propenoic acid by GC-MS analysis and a plant-bacteria degradation pathway was predicted using computational tools. Inoculation of PGP-Klebsiella sp. VITAJ23 enhanced the rate of plant growth and dye degradation.

De novo assembly and transcriptome of Pfaffia glomerata uncovers the role of photoautotrophy and the P450 family genes in 20-hydroxyecdysone production.

Pfaffia glomerata is a medically important species because it produces the phytoecdysteroid 20-hydroxyecdysone (20-E). However, there has been no ready-to-use transcriptome data available in the literature for this plant. Here, we present de novo transcriptome sequencing of RNA from P. glomerata in order to investigate the 20-E production as well as to understand the biochemical pathway of secondary metabolites in this non-model species. We then analyze the effect of photoautotrophy on the production of 20-E genes phylogenetically identified followed by expression analysis. For this, total messenger RNA (mRNA) from leaves, stems, roots, and flowers was used to construct indexed mRNA libraries. Based on the similarity searches against plant non-redundant protein database, gene ontology, and eukaryotic orthologous groups, 164,439 transcripts were annotated. In addition, the effect of photoautotrophy in two genes putatively involved in the 20-E synthesis pathway was analyzed. The Phantom gene (CYP76C), a precursor of the route, showed increased expression in P. glomerata plants cultured under photoautotrophic conditions. This was accompanied by increased production of this metabolite indicating a putative involvement in 20-E synthesis. This work reveals that several genes in the P. glomerata transcriptome are related to secondary metabolism and stresses, that genes of the P450 family participate in the 20-E biosynthesis route, and that plants cultured under photoautotrophic conditions promote an upregulated Phantom gene and enhance the productivity of 20-E. The data will be used for future investigations of the 20-E synthesis pathway in P. glomerata while offering a better understanding of the metabolism of the species.

Increased population epigenetic diversity of the clonal invasive species Alternanthera philoxeroides in response to salinity stress.

Epigenetic modification can change the pattern of gene expression without altering the underlying DNA sequence, which may be adaptive in clonal plant species. In this study, we used MSAP (methylation-sensitive amplification polymorphism) to examine epigenetic variation in Alternanthera philoxeroides, a clonal invasive species, in response to salinity stress. We found that salinity stress could significantly increase the level of epigenetic diversity within a population. This effect increased with increasing stress duration and was specific to particular genotypes. In addition, the epigenetic modification of young plants seems less sensitive to salinity than that of mature plants. This elevated epigenetic diversity in response to environmental stress may compensate for genetic impoverishment and contribute to evolutionary potential in clonal species.

Transcriptomic view of survival during early seedling growth of the extremophyte Haloxylon ammodendron.

Seedling establishment in an extreme environment requires an integrated genomic and physiological response to survive multiple abiotic stresses. The extremophyte, Haloxylon ammodendron is a pioneer species capable of colonizing temperate desert sand dunes. We investigated the induced and basal transcriptomes in H. ammodendron under water-deficit stress during early seedling establishment. We find that not only drought-responsive genes, but multiple genes in pathways associated with salt, osmotic, cold, UV, and high-light stresses were induced, suggesting an altered regulatory stress response system. Additionally, H. ammodendron exhibited enhanced biotic stress tolerance by down-regulation of genes that were generally up-regulated during pathogen entry in susceptible plants. By comparing the H. ammodendron basal transcriptome to six closely related transcriptomes in Amaranthaceae, we detected enriched basal level transcripts in H. ammodendron that shows preadaptation to abiotic stress and pathogens. We found transcripts that were generally maintained at low levels and some induced only under abiotic stress in the stress-sensitive model, Arabidopsis thaliana to be highly expressed under basal conditions in the Amaranthaceae transcriptomes including H. ammodendron. H. ammodendron shows coordinated expression of genes that regulate stress tolerance and seedling development resource allocation to support survival against multiple stresses in a sand dune dominated temperate desert environment.

Cloning and Functional Analysis of SaCLCc1, a Gene Belonging to the Chloride Channel Family (CLC), from the Halophyte Suaeda altissima (L.) Pall.

One of the genes of the CLC (Chloride Channel) family, SaCLCc1, from the halophyte Suaeda altissima (L.) Pall. was cloned. To investigate the function of SaCLCc1, it was expressed in the S. cerevisiae deletion mutant Δgef1::LEU2 for the only gene of the CLC family in this organism. The growth of the transformed SaCLCc1-expressing mutant Δgef1 was restored when cells were grown in Fe2+-deficient YPEG medium, in minimal synthetic media SD and SR (pH 7.0), and in rich YPD medium containing Mn2+. The complementation of the Δgef1 mutant phenotype with the SaClCc1 gene indicates the involvement of the SaClCc1 protein in the transport of Cl- ions.

Bona fide choline monoxygenases evolved in Amaranthaceae plants from oxygenases of unknown function: Evidence from phylogenetics, homology modeling and docking studies.

Few land plants can synthesize and accumulate the osmoprotectant glycine betaine (GB) even though this metabolic trait has major adaptive importance given the prevalence of drought, hypersaline soils or cold. GB is synthesized from choline in two reactions catalyzed by choline monooxygenases (CMOs) and enzymes of the family 10 of aldehyde dehydrogenases (ALDH10s) that gained betaine aldehyde dehydrogenase activity (BADH). Homolog genes encoding CMO and ALDH10 enzymes are present in all known land plant genomes, but since GB-non-accumulators plants lack the BADH-type ALDH10 isozyme, they would be expected to also lack the CMO activity to avoid accumulation of the toxic betaine aldehyde. To explore CMOs substrate specificity, we performed amino acid sequence alignments, phylogenetic analysis, homology modeling and docking simulations. We found that plant CMOs form a monophyletic subfamily within the Rieske/mononuclear non-heme oxygenases family with two clades: CMO1 and CMO2, the latter diverging from CMO1 after gene duplication. CMO1 enzymes are present in all plants; CMO2s only in the Amaranthaceae high-GB-accumulators plants. CMO2s, and particularly their mononuclear non-heme iron domain where the active site is located, evolved at a faster rate than CMO1s, which suggests positive selection. The homology model and docking simulations of the spinach CMO2 enzyme showed at the active site three aromatic residues forming a box with which the trimethylammonium group of choline could interact through cation-π interactions, and a glutamate, which also may interact with the trimethylammonium group through a charge-charge interaction. The aromatic box and the carboxylate have been shown to be critical for choline binding in other proteins. Interestingly, these residues are conserved in CMO2 proteins but not in CMO1 proteins, where two of these aromatic residues are leucine and the glutamate is asparagine. These findings reinforce our proposal that the CMO1s physiological substrate is not choline but a still unknown metabolite.

Organization and evolution of two repetitive sequences, 18-24J and 12-13P, in the genome of Chenopodium (Amaranthaceae).

The abundance and chromosomal organization of two repetitive sequences named 12-13P and 18-24J were analyzed in 24 diploid and nine polyploid species of Chenopodium s.l., with special attention to Chenopodium s.s. Both sequences were predominantly present in species of Chenopodium s.s.; however, differences in the amplification levels were observed among the species. The 12-13P repeat was highly amplified in all of the analyzed Eurasian species, whereas the American diploids showed a marked variation in the amplification levels. The 12-13P repeat contains a tandemly arranged 40 bp minisatellite element forming a large proportion of the genome of Chenopodium (up to 3.5%). FISH revealed its localization to the pericentromeric regions of the chromosomes. The chromosomal distribution of 12-13P delivered additional chromosomal marker for B-genome diploids. The 18-24J repeat showed a dispersed organization in all of the chromosomes of the analyzed diploid species and the Eurasian tetraploids. In the American allotetraploids (C. quinoa, C. berlandieri) and Eurasian allohexaploids (e.g., C. album) very intense hybridization signals of 18-24J were observed only on 18 chromosomes that belong to the B subgenome of these polyploids. Combined cytogenetic and molecular analyses suggests that reorganization of these two repeats accompanied the diversification and speciation of diploid (especially A genome) and polyploid species of Chenopodium s.s.

Evolutionary diversification of the African achyranthoid clade (Amaranthaceae) in the context of sterile flower evolution and epizoochory.

Background and Aims: Many African genera of the Amaranthaceae exhibit unique inflorescences that include sterile flowers modified to hooks or spines. Considering that the abundance of large terrestrial herbivores increased on the African continent with the expansion of grassland and savannah ecosystems, modified sterile flowers could have been an innovation that boosted the diversification of an African achyranthoid clade of Amaranthaceae, with large animals serving dispersal. Methods: We generated an extensively sampled phylogeny comprising 26 of the 31 achyranthoid genera as well as representatives of all other lineages of Amaranthaceae. Phylogenetic tree inference employed four genomic regions, using parsimony, likelihood and Bayesian inference methods. We estimated divergence times, evaluated trait-dependant changes and species diversification rates using state-dependent speciation and extinction models, and reconstructed ancestral character states for modified sterile flowers. Key Results: The achyranthoids were found to be a major clade of the Amaranthaceae, comprising mostly African members. Phylogenetic relationships within this clade were well resolved and supported two main subclades. Several genera were found to be polyphyletic. Our results indicate that the achyranthoids started to diversify ~28 million years ago, and that modified sterile flowers evolved multiple times. An asymmetry in transition rates towards the gain of sterile flowers was observed, whereas no trait-dependent increase in species diversification rates was detected. Bayesian rate heterogeneity analyses indicated that the achyranthoids diversified without significant rate shifts. Conclusions: The accumulation of modified sterile flowers within achyranthoids appears to result from the higher transition rates in favour of modified sterile flowers. Multiple gains suggest an adaptive value for this trait. However, epizoochory does not appear to fuel species diversification, possibly due to extensive gene flow through regularly migrating mammals, which limits the possibility of speciation by isolation.